Signaling by the Wnt family of secreted proteins regulates many aspects of embryogenesis and homeostasis. Deregulated Wnt signaling is implicated in human diseases such as cancer, osteoporosis and degenerative disorders. The study of Wnt signaling thus has critical importance for understanding basic biology and human health. In the canonical Wnt pathway that regulates the stability of the transcriptional coactivator 2-catenin, two types of transmembrane receptors that constitute the Wnt receptor complex are involved. One is a member of the Frizzled (Fz) family of serpentine receptors, and the other is a single-pass transmembrane receptor of the LDL receptor related protein (LRP) family, LRP5 or LRP6. We have shown that Wnt induces LRP6 phosphorylation/activation, which recruits the Axin scaffolding protein that is essential for the regulation of 2-catenin stability. But how LRP6 phoshorylation and activation and the recruitment of Axin lead to 2- catenin stabilization has been poorly understood, and is the focus of this application. Aim 1 is to study LRP6 phosphorylation and signaling. We have generated Lrp5-/-;Lrp6-/- double knockout (LrpDKO) mouse embryonic fibroblast cells. These LrpDKO cells provide an ideal genetic plus biochemical system for in-depth investigation of LRP6 phosphorylation and signaling. We will re-express the wild type and various LRP6 mutants in Lrp6DKO cells to define the initiating step in LRP6 phosphorylation, the function and relationship among distinct phosphorylation motifs, and their potential roles in LRP6 signaling and in the so-called LRP6 "signalosome" formation. Aim 2 is to study Wnt regulation of Axin dephosphorylation. Axin is a pivotal scaffolding protein for 2-catenin phosphorylation/degradation and LRP6 phosphorylation/activation, and Axin interaction with phosphorylated LRP6 is a key step in 2-catenin stabilization. We have generated specific antibodies for phoshorylated Axin, and show that Wnt promotes Axin dephosphorylation via protein phosphatase 1 (PP1). We found that this is likely mediated through phosphorylation of a PP1 regulatory protein, a previously unsuspected component in Wnt signaling. We will study Axin dephosphorylation and its role in mediating Wnt-regulated Xenopus embryonic patterning. Aim 3 is to study Axin-LRP6 interaction. We will establish a new type of phosphorylation-regulated protein interaction between Axin and LRP6 and uncover how Axin phosphorylation controls its interactions with other partners such as LRP6 via a novel intra- molecular and auto-regulatory mechanism. We will present a cohesive working model that links LRP6 activation to 2-catenin stabilization through regulation of Axin phosphorylation-dephosphorylation. Aim 4 is to study the mechanism by which Wnt activates PP1 dephosphorylation of Axin. We will study how LRP6 regulates PP1 activation using LrpDKO cells, and we are particularly interested in the identification of the kinase (or kinases) that mediates Wnt activation of PP1. These studies will likely provide a comprehensive understanding of Wnt signaling from the Wnt receptor to 2-catenin stabilization. PUBLIC HEALTH RELEVANCE: Cell-to-cell communication is vital for human embryonic development and tissue homeostasis, and is mediated by key "signal transduction" pathways. Defects in these pathways cause human cancer. This proposal aims to understand the molecular nature of one of these signaling pathways, the so-called Wnt pathway, in animal development and human cells. Our study will provide better understanding of human embryonic development and pathogenesis of cancer.